Malkki M, Laakso M, Deeb S S
Department of Medicine, University of Washington, Seattle 98195, USA.
Diabetologia. 1997 Dec;40(12):1461-9. doi: 10.1007/s001250050850.
Hexokinase II (HKII) plays an important role in facilitating glucose uptake by skeletal muscle, heart, and adipose tissue in response to insulin. We have cloned and sequenced the proximal promoter region of the human HKII gene, determined the transcription start sites and screened the 2.0 kb of the proximal 5' flanking region for variants in non-insulin-dependent diabetic patients and control subjects. We found three variants in this region, one in the 5' untranslated region (G-->C at +217) and two in the promoter region (T-->G at -1043 and G-->A at -1159). The allele frequencies of these variants did not differ between the diabetic and control subjects and these variants are not associated with insulin resistance. Various segments of the human HKII promoter were tested for driving expression of the luciferase reporter gene. The proximal 500 bp and 400 bp of the promoter were sufficient to drive maximal activity in adipocyte (3T3F442A) and myocyte (C2C12F3) cell lines, respectively. This region of the promoter is GC-rich and contains eight consensus binding sites for the transcription factor Sp-1, five for AP-2, two putative response elements for each of insulin and cyclic AMP. The proximal 175 bases of the promoter retained only 7-15% of maximal activity. Sequence elements located between positions -304 and -215 accounted for approximately 80% of the basal HKII promoter. In addition, the region between -215 and -184 contains a negative regulatory element for expression in 3T3F442A but not in C2C12F3 cells.
己糖激酶II（HKII）在促进骨骼肌、心脏和脂肪组织响应胰岛素摄取葡萄糖的过程中发挥着重要作用。我们克隆并测序了人类HKII基因的近端启动子区域，确定了转录起始位点，并在非胰岛素依赖型糖尿病患者和对照受试者中筛查了近端5'侧翼区域2.0 kb的变异情况。我们在该区域发现了三个变异，一个位于5'非翻译区（+217处G→C），两个位于启动子区域（-1043处T→G和-1159处G→A）。这些变异的等位基因频率在糖尿病患者和对照受试者之间没有差异，且这些变异与胰岛素抵抗无关。对人类HKII启动子的各个片段进行了测试，以驱动荧光素酶报告基因的表达。启动子的近端500 bp和400 bp分别足以在脂肪细胞（3T3F442A）和肌细胞（C2C12F3）细胞系中驱动最大活性。启动子的该区域富含GC，包含八个转录因子Sp-1的共有结合位点、五个AP-2的结合位点、两个胰岛素和环磷酸腺苷各自的假定反应元件。启动子的近端175个碱基仅保留了最大活性的7-15%。位于-304和-215位之间的序列元件约占HKII启动子基础活性的80%。此外，-215和-184之间的区域在3T3F442A细胞中含有一个表达的负调控元件，但在C2C12F3细胞中没有。
